Efficient Differentiation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Mesenchymal Progenitors Into Adipocytes and Osteoblasts

Human induced pluripotent stem cells (hiPSCs) hold immense promise in regenerative medicine as they can differentiate into various cell lineages, including adipocytes, osteoblasts, and chondrocytes. Precisely guiding hiPSC-derived mesenchymal progenitor cells (iMSCs) towards specific differentiation pathways is crucial for harnessing their therapeutic potential in tissue engineering, disease modeling, and regenerative therapies. To achieve this, we present a comprehensive and reproducible protocol for effectively differentiating iMSCs into adipocytes and osteoblasts. The differentiation process entails culturing iMSCs in tailored media supplemented with specific growth factors, which act as cues to initiate adipogenic or osteogenic commitment. Our protocol provides step-by-step guidelines for achieving adipocyte and osteoblast differentiation, ensuring the generation of mature and functional cells. To validate the success of differentiation, key assessment criteria are employed. For adipogenesis, the presence of characteristic lipid droplets within the iMSC-derived cells is considered indicative of successful differentiation. Meanwhile, Alizarin Red staining serves as a marker for the osteogenic differentiation, confirming the formation of mineralized nodules. Importantly, the described method stands out due to its simplicity, eliminating the need for specialized equipment, expensive materials, or complex reagents. Its ease of implementation offers an attractive advantage for researchers seeking robust and cost-effective approaches to derive adipocytes and osteoblasts from iMSCs. Overall, this protocol establishes a foundation for exploring the therapeutic potential of hiPSC-derived cells and advancing the field of regenerative medicine. Key features • iMSC derivation in this protocol uses embryonic body formation technique. • Adipogenesis and osteogenesis protocols were optimized for human iPSC-derived iMSCs. • Derivation of iMSC from hiPSC was developed in a feeder-free culture condition. • This protocol does not include human iPSC reprogramming strategies.

This protocol was used in: eLife (2023), DOI: 10.7554/eLife.83138 Human induced pluripotent stem cells (hiPSCs) hold immense promise in regenerative medicine as they can differentiate into various cell lineages, including adipocytes, osteoblasts, and chondrocytes.Precisely guiding hiPSCderived mesenchymal progenitor cells (iMSCs) towards specific differentiation pathways is crucial for harnessing their therapeutic potential in tissue engineering, disease modeling, and regenerative therapies.To achieve this, we present a comprehensive and reproducible protocol for effectively differentiating iMSCs into adipocytes and osteoblasts.The differentiation process entails culturing iMSCs in tailored media supplemented with specific growth factors, which act as cues to initiate adipogenic or osteogenic commitment.Our protocol provides step-by-step guidelines for achieving adipocyte and osteoblast differentiation, ensuring the generation of mature and functional cells.To validate the success of differentiation, key assessment criteria are employed.For adipogenesis, the presence of characteristic lipid droplets within the iMSC-derived cells is considered indicative of successful differentiation.Meanwhile, Alizarin Red staining serves as a marker for the osteogenic differentiation, confirming the formation of mineralized nodules.Importantly, the described method stands out due to its simplicity, eliminating the need for specialized equipment, expensive materials, or complex reagents.Its ease of implementation offers an attractive advantage for researchers seeking robust and cost-effective approaches to derive adipocytes and osteoblasts from iMSCs.Overall, this protocol establishes a foundation for exploring the therapeutic potential of hiPSC-derived cells and advancing the field of regenerative medicine.Laboratory supplies Cite as: Diaz-Hernandez, M. E. et al. (2023).Efficient Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) Derived Mesenchymal Progenitors into Adipocytes and Osteoblasts.Bio-protocol 13(22): e4885.Sterile filter with 0.2 μM pore microfilter.Aliquots are stored at -20 °C.

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Cite as: Diaz-Hernandez, M. E. et al. (2023).Efficient Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) Derived Mesenchymal Progenitors into Adipocytes and Osteoblasts.Bio-protocol 13(22): e4885.DOI: 10.21769/BioProtoc.4885.Nov 20, 2023 Calculate the Collagen 1 rat tail volume needed: 20 μg/mL collagen × final volume = stock collagen I rat tail (μg/mL) Calculate the volume of 20 mM acetic acid needed: Final volume needed-Volume of Collagen1 stock.100μL of the solution is added to each well of the 24-well plate.Add the solution to the plates.Incubate at room temperature for 1 h.Do not freeze the Collagen 1 rat tail.

10 mM dexamethasone solution (F.W: 392.4 g/mol) (stock solution)
As a general guide, small molecules in DMSO solution are recommended to be stored in small aliquots at -20 °C.Aliquot the solutions into working volumes to avoid freeze-thaw cycles.Protect from prolonged exposure to light.

4. 10 mM indomethacin (F.W: 357.9 g/mol) (100× stock solution)
It is recommended to store DMSO aliquots at -20 °C.Aliquot the solutions into working volumes to avoid freeze-thaw cycles.Protect from prolonged exposure to light.
3% Oil red working solution: mix three parts of 3% oil red stock solution and two parts of distilled water and allow to sit for 10 min.Final solution is Whatman paper filtered.This solution is stable for 2 h.3%Oil red stock solution is stable for one year.Dissolve PFA in a heating and stirring block (60 °C) and in a chemical hood.Adjust to 7.4 pH with 1 M HCl.Adjust to 100 mL final volume.Aliquots can be stored at -20 °C.